The determination, proliferation and differentiation of muscle cells during development in both vertebrates and invertebrates depend upon the complex regulated expression, dimerization and function of the MyoD family of basic helix-loop-helix proteins. We have continued to study the role of the MyoD related proteins during vertebrate and Drosophila myogenesis. In the vertebrates, the MyoD family of muscle gene regulatory proteins or MRFs, were thought to directly auto regulate their expression levels through MRF binding sites in the promoters of these genes. However, we have demonstrated that this pathway is indirect, at least for avian MyoD (CMD1), and does not involve any presently known muscle-specific transcription factors. In Drosophila we have shown that the single MyoD gene, nau is not auto regulated but expression depends upon an earlier mesodermal transcription factor, DMEF2. The single DMEF2 gene encodes a MADS domain containing SRF homologue essential for muscle formation in Drosophila. DMEF2 is expressed just after the onset of twist expression, the key mesodermal determination gene in flies. Our studies indicate that, in addition to DMEF2, twist also regulates nau in cultured Drosophila cells, suggesting twist is epistatic to nau and is a possible regulator of nau during embryogenesis at the earliest point of mesoderm formation. The MyoD family of proteins are phosphoproteins. Two adjacent serine residues outside the HLH domain in the carboxy terminal portion of vertebrate MyoD regulate the formation of the homodimer in vitro and in vivo. This site and its function are conserved in nau. Phosphorylation of this site also regulates a transcriptional activation domain and may be responsive to signal transduction pathways that modulate MyoD function. We have demonstrated the importance of dimerization specificity among the MyoD/E-protein family members by using a newly developed competitive EMSA, demonstrating the heterodimer dependent myogenic conversion of nonmuscle cells from both vertebrates and Drosophila, and conducting the rescue of a MyoD lethal mutation in C. elegans with Drosophila MyoD, a predicted heterodimer partner from our studies on dimerization specificity. MyoD is not only a transcription factor but also appears to play a role in the decision to exit the cell cycle during differentiation. Preliminary experiments indicate that MyoD can regulate CDK function in a concentration dependent manner, inhibiting the phosphorylation of Rb. The fragment of MyoD containing the CDK binding site can itself inhibit cell growth.